Method of deodorizing amine-containing material utilizing cis-succinic acid of gauche form



g- 1969 TADATAKA HARA 3, 9,

METHOD OF DEODORIZING AMINE-CONTAINING MATERIAL UTILIZING CIS-SUCCINIC ACID 0F GAUCHE FORM Original Filed Nov. 15. 1966 4 Sheets-Sheet 1 QNQQfiQQQQQtQxQmmmhwmw ub u \m I! l l I Bu mtmEa T L BQ Q GQ I @55 3 cmmg e o i vm o urmb E 44mm,

mvsmon BY NM 4 ATTORNEY g" 5, 1969 TADATAKA HARA 3, 59,

METHOD OF DEODORIZING AMINE-CONTAINING MATERIAL UTILIZING CIS-SUCCINIC ACID OF GAUCHE FORM Original Filed Nov. 15, 1966 4 Sheets-Sheet 2 Fuman'c acid (5/ ml Presenf subsfvnc MOO/ml Succ/h/c 00/0 0 (6/074 A l l j l l I l 200 2/0 220 Wdve/engfh my) INVENTOR ATTORNEYS 7 Original Filed Nov. 15. 1966 Aug. 5, 1969 TADATAKA HARA 3,459,491

METHOD OF DEODORIZING AMINE-CONTAINING MATERIAL UTILIZING CIS-SUCCINIC ACID OF GAUCHE FORM 4 Sheets-Sheet 3 F/6. 3 Press/7f subsfance (nu/0H Sum/M00010 (nu/0i 2000 /000 Wave number 6/14 /6 uo ssywsuw INVENTOR BYLQMzMZ MM ATTORNEYS 8- 5, 6 TADATAKA HARA 3,459,491

METHOD OF DEODORIZING AMINE-GONTAINING MATERIAL UTILIZING CIS-SUCCINIG ACID 0F GAUCHE FORM Original Filed Nov. 15. 1966 4 Sheets-Sheet 4- United States Pat eg METHOD OF DEODORIZING AMINE-CONTADI- ING MATERIAL UTILIZING ClS-SUCCINIC ACID F GAUCHE FORM Tadataka Piara, Tokyo, Japan, assignor to Kaken Kagaku Kabushiki Kaisha, Tokyo, Japan, a corporation of Ja an Application Nov. 15, 1966, Ser. No. 594,602, now Patent N 0. 3,352,759, dated Nov. 14, 1967, which is a continuation-in-part of application Ser. No. 860,227, Dec. 17, 1959. Divided and this application Aug. 3, 1967, Ser. No. 669,331

Claims priority, application Japan, Jan. 14, 1959, 34/ 534; June 29, 1959, 34/20,893 Int. Cl. A611 3/00, 13/00 11.8. Cl. 21-55 3 Claims This application is a division of Ser. No. 594,602, now US. Patent 3,352,759, which is in turn a continuation-inpart of Ser. No. 860,227, filed on Dec. 17, 1959, and now abandoned.

This invention relates to a novel and useful compound called cis-succinic acid of Gauche form and particularly concerns a method for deodorizing amine-containing materials by contacting them with the said cis-succinic acid of Gauche form.

Heretofore, the existence of Gauche form isomers of succinic acid have been assumed theoretically, but not confirmed. According to the studies of this inventor, it was found that commercially available succinic acid synthesized by chemical methods is mostly of trans-form and contains a small amount of cis-form. These two may be separated by their solubility difference in ether. It is also pointed out that there are different shapes of crystals in succinic acid, each being distinguishable under micro scopical examination, and these are classified into two types depending on their respective shape at room temperature, i.e., ot-type for needle shape and fi-type for hexagonal board or short cylinder. These different crystal types are known as polymorphism and usually has a distinct transition point at which one crystal type can be transformed into the other type. Ordinarily, succinic acid is believed to have no transition point, but the present cissuccinic acid of Gauche form possesses a distinct transition point. As described hereinabove, commercial succinic acid is the mixture of trans-form and a small amount of cis-form (ix-type and ,B-type). The B-type crystal is stable at relatively lower temperature while the a-type crystal is stable at relatively higher temperature.

Dupr 1a Tour reported in Compt. Rend, vol. 193, page 180 (1931), that when ,G-type succinic acid is heated at 138 C., this crystal is converted to u-type and at the same time cis-form acid is produced, and therefore the transition point of succinic acid is 138 C. However, the inventor examined the properties of commercially available succinic acid and found that the trans-form succinic acid cannot be converted to the cis-form by mere heating, but can be converted to a-type from fl-type when it is first molten at 186 C. and subsequently cooled to precipitate crystals, accordingly, the distinct transition point cannot be observed. On the other hand, it was also found that the pure cis-form succinic acid possesses a considerably clear transition point from ]3-- to o't-type crystals.

The following important facts were also found by the inventor. That is, the crystal shape of this substance is in general hexagonal board or short cylinder at a room temperature. Upon heating, it is completely sublimed at l47-155 C., but its original shape is still maintained after said sublimation. Continuing the heating, the compound, however, suddenly changes into long cylindrical shape at 15 6157 C. From these findings, the inventor ratiocinated that this long cylindrical shape must be the a-type succinic acid.

Patented Aug. 5, 1969 In the chemical field, it is well known that some chemicals possess optical activity even if they have no asymmetrical carbon atom. For example, dichloroethane having the formula (trans) carries no asymmetrical carbon atom, but the intramolecular rotation therein is somewhat restricted by the presence of two chlorine atoms and the free rotation is settled down at positions where the rotation potential is minimal. Thus, the trans-dichloroethane possesses the rotational isomers having one CH C1 turned by i around the axis CC. These rotational isomers are often called Gauche form isomers.

Since succinic acid is represented by either of the following formulae quite similar to that of dichloroethane.

sis-succinic acid trans-succinic acid Such Gauche form isomers are assumed theoretically, but they have not been confirmed up to the time.

The present inventor has discovered that a Gauche form isomer closely related to the cis-type succinic acid can be prepared together with cis-succinic acid by fermentation with a strain of Saccharomyces genus. These two, i.e., cis-type and now Gauche form, are obtained as a mixture, but can hardly be separated from each other because of the similarity in their respective physical and chemical properties. However, this product apparently differs from the existing succinic acids, i.e., trans-form, in many respects as described below, and possesses an outstanding characteristic activity to remove the offensive odor derived from amines. The terms cis-succinic acid of Gauche form used herein refers to the product obtained by the present method.

The object of the invention is to provide a method for deodorizing amine-containing materials by contacting them with the said cis-succinic acid of Gauche form. Other objects, features, capabilities and advantages comprehended by the present invention will be apparent from the description and claims which follow.

Referring to the drawings:

FIG. 1 is a curve of the titration of fl-cis-succinic acid of Gauche form, with N/ 10 sodium hydroxide aqueous solution;

FIG. 2 is a curve of the ultra-violet spectrum of p-cissuccinic acid of Gauche form;

FIG. 3 is a curve of the infra-red spectrum of p-cissuccinic acid of Gauche form; and

FIG. 4 are crystals of [3-cis-succinic acid of auche form, and

FIG. 5 is crystals of oz-CiS-SuCciniC acid of Gauche form.

This new compound, cis-succinic acid of Gauche form, can be formed by growing the microorganism, Saccharomyces steineri var. hara in a suitable nutrient medium under appropriate stationary or submerged aerobic (viz. shaken) conditions. This strain, which we have isolated from home-made fermented grape juice, and which we designate as Saccharomyces steineri var. hara, is so similar in cultural characteristics to a strain of Saccharomyces sreineri with minor exceptions that I consider it to be belonging to the same species with Saccharomyces steineri. A culture of the living microorganism has been deposited with and is available from the American Type 3 Culture Collection; it has been designated as ATTC No. 13668.

The strain was isolated from the fermented grape juice, and the method of isolation is as follows:

Well washed grape was milled and then placed in a sterile flask with cotton-seal. Fermentation was carried out at 25 C. for 3 to 4 days. During this period, the flask was stirred by shaking twice a day. At the end of fermentation, a fermentation liquor was taken by a platinum loop and were inoculated to malt agar plate. The plate was incubated at 25 C. for 48 hours. On the plate surface, the pale yellow, creamy colonies of the desired Saccharomyces steineri var. ham and the white, filmy wrinkled colonies were formed, the former colonies being collected to provide the desired microorganism.

The present invention will be described with particular reference to the above-mentioned strain of the organism, but it is to be understood that the fermentative processes of this invention embrace not only the use of Saccharomyccs sleinel'i var. ham but also other Gauche form cis-succinic acid-producing strains of Saccharomyces genus, such strains being readily produced and isolated by routinely applied isolation and strain-modification methods which include selection of cultured organisms, and exposure of organisms to modifying means such as X-ray, ultraviolet light, and chemical agents, for example, nitrogen mustards. Illustrative examples of other Gauche form cis-succinic acid-producing strains are Saccharo- 4- white colored deposits in the culture medium. No film formation is observed.

GROWTH ON MALT AGAR The growth of the strain on malt agar is just equal to that on malt juice. There is no significant difference in shape and size of cells. The colonies of the strain are round, semiglossy, light yellow-white color creamy.

After cultivations of the slant culture for one month, the colonies are found to be yellowish-white colored, creamy soft semi-glossy and smooth, not wrinkled.

Slide culture: Pseudomycelium is not found. (An original strain of Sacclz. steineri, and strains of Sacch. cereviciae, Sacch. cereviciae var. ellipsoidcus and Sacclz. willianus are different from each other in their shape, and therefore the present strain can be macroscopically distinguished from them.)

Sporulation: Spores are readily formed on malt agar. Usually an ascus contains four round escospores. In this point the present strain is differed from an original strain of Sacch. steineri.

FERMENTATION OF VARIOUS SUGARS The present strain Saccharomyces steineri var. ham

shows the following fermentative activity to various sugars.

. Glucose myces cerevzszae, Saccharomyces cerevzslae var. ellzpsoz- Galactose deus, and Saccharomyces wzllzanus. 30 Maltese Saccharomyces steineri var. Izara (ATCC No. 13668) n Saccharose is characterized by the physical, cultural, and physiolo ical tests set forth in the followin table and para- Lactose b b Raffinose graphs.

GROWTH ON MALT JUICE ASSIMILATION OF POTASSIUM NITRATE Potassium nitrate is not assimilated by this strain.

DECOMPOSITION OF ARBUTIN This strain does not decompose arbutin. The comparative table with respect to the present strain and other assembling known yeast is as follows:

Sediment forma- Ring Shape of the cell Size of the cell, ,1: tion formation Pseudomycelium Sporulation The present strain Oval to elongate +(thin or no) +trce-like... Round, mostly 4/aseus. Saccharomyces stei'ncri Oval to long-oval 3-8 X 5-14 +(thin) do Round 2-4/ascus mostly 2. 2.2-5.5 x 6-14-33 Round or oval Sacch. wzlltanus Oval to elongate 3-6.5 x 6. 5-14-23 +tree-formatlon- 1-4/asc.us

4-7 x 8-15-22 3.5-9.5 x 6-14-20 Sacch. care. var. clhps0idcus Oval to long-0val 2. 5-6 x 3. 5-13 +(poo1) Do.

3-7.5 2 1 5-14 5-10 x 6-12 (one third of Round or alightly Sacch. ccrcvzszae Round, short-oval or oval. 3-7 82: 4. 5-10 :l; the strain). oval 1-4/ascus. 3.5- x 5-15 Sacch. pastorz'anus Ovla1, elongate or sausage- 2.5-4 x 7-26 +tree-formation. Round 1-4/ascus.

s rapes. 5-10 x 5-13 usually, +(elon- D0. Sacch. carlsbergamszs Round, short oval or oval. 5 x 515' 5 gate cens) Sacch. ltetcrogemcous Oval to long-oval 3-6 x 6-12 +t1ee-like Do. Saccl1.1talicus d0 4-8 x 5-15 +(primitlve) Do.

Fermentation Sugar assimilation Glucose Maltose Galactose Lactose Saccha- Raffinose Glu- Mal- Galactose Lac- Saecharose cose tose se rose The present strain Saccharomyces steineri. i i i i Sacch. sillitmus +(weak) +14 Sacch. cere. var. ellipsoz'deus.-. Sacch. cerevisiae +(weak or +(weak) Sacch. pastorianus... +36 Sacch. carlsbergemszs +(complete) Sacch. heterogenicu3 Sacch. italicus l weak Assimilation of S llttin of Potassium nitrate Ethanol as sole source of carbon ar butin g The present strain Absent N0 g owth. Absent. Saccharomyces steineri. d0 Do. Sacch. williamts N0 growth or weak growth Do. Sacch. cere. var. ellipsoildus Usually no grow h weak or normal growth Do. Sacch. cerem'siae .do. Usually 110 growth occasionally weak or normal growth Do. Sacch. pastorianus-.. N0 g ow h Do. Sacch. carlsbergem'sis do do. Do. Sacch. heterogem'cus do Do. Sacch. italicus do Do.

lhe classification and diagnosis are performed based upon The Yeasts" a toxonomic study by J. Lodder and N. J. W. Kroger, Van Rij North- Holland Publishing 00., Amsterdam (1952).

As the result of the tests stated, the present strain has been identified as a new strain of Saccharomyces steineri. Analogous strains include Saach. cerevioiae, Sacch. steineri, Sacch. ellipsoideus, Sacch. willianuis, etc. In the fermentative activity for rafiinose, this strain is similar to Table 2 shows the result of fermentation tests using standing culture at 27 C. for 14 days. A series of these tests was carried out for determining the usefulness of various culture media.

Saach. steineri, and diifered from the strains of other TABLE 2 species. This strain forms two ascopores in ascus, while B f After the original strain of Saach. steineri usually forms four cultlvatwn cultlvatwn ascopores. D d sugar Sugar In the present invention, grape juice or malt juice may gg g" ggg: figg: be used as culture medium. Malt uice is rather preferred Culture medulm effect Percent P percent PH because it is readily commercially available. Of course Gmwiuice 1570 M M other culture media include cane sugar uice, Henne- 1 15.0 5.0 9.0 4.0 bergs synthetic medium, etc. can be used to produce 19- i edium zl syn e10 cis-succinic acid of Gauche form. K21 322 i 12.3 5.8 3.8 Table 1 shows the result of fermentation tests using g gf igg jj 4 7.248 shaking culture at 27 C. for 72 hours. A series of these 233 t atgfl i 12.8 7 .8 fig 2.3 tests was carried out for determining the usefulness of f iff: 1 1 various culture media.

1 Nitrogen source. TABLE 1 Before After With respect to cultivation method of the present incultlvatmn cumvamn vention, any of standing culture, shaking culture, sub- Sugar Sugar merged aerated culture, etc., may be employed to form P 1 ,B-cis-succinic acid of Gauche form. However, when ization tamed, tamed, Culture medium effect percent pH percent pH aeroblc cultivation such as shaking culture and submerged er u Grapejuice 15 3.5 M 25 a ated c lt ure 1s employed, the time requlred for cu t1 Malt juice 15 5.0 40 3.2 vatlon 1s hlghly mmunized. ACCOIdlIlglY, such methods f fi ff Synthetic are more preferably used for the purpose of the inven- Peptone 15 7.0 as 4.0 tion, as compared with stationary cultivation methods. fi ggi fif g-g g-g Under aerobic conditions, the cultivation may be successfioniumnitrate: 15 7:0 9:7 513 fully carried out at about 20 to 30 C. for about 72-96 Ammonium i 15 hours When stationary conditions are applied to cultiva- 15 7.0 12.4 6.4-6.2 Sodium nitrate tron, it should be carried out at about 20 to 30 C. for Nitrogen source. b t 14 21 d TABLE 3.STANDING CULTURE TEST 0 day 7 days 14 days 21 days Tern Sugar, Sugar, Sugar, Sugar, Medium 0. percent pH percent pH percent pH percent pH 27-30 14.6 3.5 +7.9 3.0 +6.4 2.7 ++4.s 2.5 Grape juice..-" 15-20 14.6 3.5 8.2 3.2 +7.2 3.0 +6.6 2.8 5 14.6 3.5 -a.4 3.4 +7.6 3.2 +7.4 3.0 27-30 13.2 5.0 -8.6 3.4 +5.4 3.0 ++3.2 2.7 Malt juice 15-20 13.2 5.0 -9.6 3.3 +7.4 3.2 +4.0 2.9 5 13.2 5.0 -11.4 4.6 -10.0 4.0 +8.6 3.3

TABLE 4.SHAKING CULTURE TEST 0 hr. 24 hrs. 43 hrs. 72 hrs.

Temp, Sugar, Sugar, Sugar, Sugar, Medium 0. percent pH percent pH percent pH percent pH Grape juice..-" 27 15.0 3.5 2.5 ++5.0 2.5 ++4.e 2.5 Malt juice 27 15.0 5.0 +5.4 3.7 ++5.2 3.4 ++4.2 3.

TABLE 5.COMPARATIVE TEST FOR SHAKING CULTURE USING VARIOUS STRAINS hr. 12 hrs. 24 hrs. 48 hrs. 72 hrs.

Sugar, Sugar, Sugar, Sugar, Sugar,

Strain percent pH percent pH percent pH percent pH percent pH Sacch. steineri var. ham 13 5.0 -12. 2 4. 6 3:5. 4 3. 7 -|-+3.1 3. 1 ++2. 8 3.0 Sacch. steineri 13 5. 0 12. 9 4. 8 i6. 4. 0 +4. 0 3. 8 ++4. 0 3. 8 Sacch. c. v. ellipsoids 13 5.0 -12.8 4. 8 i6. 6 3. 9 +4. 6 3. 9 ++4. o 3. 8 Sacch. cereviciac 13 5. 0 12. 9 4. 9 -10. 1 4.0 10.0 4.0 :l=10. 0 4. 2

In Tables 3 and 4, both tests were carried out using a strain Saccharomyces steineri var. lzara (ATCC No. 13668). In Table 5, the tests were carried out at 27 C., using malt juice medium. In each of the above tables, signs (1:), and indicate the degree of deodorization effect.

In carrying out the invention in effect, the culture solution obtained by cultivation stated above is extracted with ether, and then the ether is removed from the extract. The remaining part is further concentrated to yield crude crystal of Gauche form ,B-cis-succinic acid. The crude product can be recrystallized form hot ether to give pure crystal.

Thus, the Gauche form succinic acid can be prepared economically by the fermentation with a strain Saccharomyces steineri var. hara (ATCC No. 13668) and by an ether extraction of the culture solution obtained. The inventor has further discovered that the above said ether extract contains a useful enzyme like yellow substance, that is, the water solution of commercially obtainable succinic acid and maleic acid were changed to the fi-cis-succinic acid of Gauche form by contacting with above yellow substance under light shielding conditions.

Firstly an enzyme like substance containing liquor is prepared by evaporating the solvent from the above said ether extract. A mixture of equal amounts of known, commercially available succinic acid (optical isomer) and maleic acid or maleic anhydride is dissolved in twice amount of distilled water under heating. After cooling to a temperature below 50 C., the resulting solution is combined with the enzyme like substance containing liquor under day-light shielding conditions. The resulting mixture is concentrated under reduced pressure to yield crude crystal of ,B-cis-succinic acid of Gauche form.

According to the method described herein, almost 90% of the total weight of the succinic acid and maleic acid employed can be converted to the Gauche form succinic acid.

With respect to the production of ,B-cis-succinic acid of Gauche form, typical procedures used for carrying out the invention will be described in the following paragraphs.

CULTIVATION OF SACCH. STEINERI VAR. HARA Two hundreds milliliters of malt juice was charged into a 500 cc.-volume Sakaguchi flask and then sterilized with heat at 120 C. for 20 minutes. After cooling, a strain Sacch. stez'neri var. hara was planted to the flask. Before cultivation, malt juice preferrably had a pH of 4.6 to 5.6 and a sugar content of 15 to 17%. Cultivation temperature was 25i5 C. and cultivation period was 72 to 120 hours. During cultivation, the flask was shaken by means of a reciprocating type shaking machine which was reciprocated 120 times per minute with an amplitude of 8 cm. After cultivation, the culture solution was found to have a pH of 2.8 to 3.2 and a sugar content of 3 to 4%.

EXTRACTION OF B-CIS-SUCCINIC ACID OF GAUCHE FORM FROM CULTURE SOLUTION From the culture solution obtained by cultivation stated, mycelia were removed by filtration. The filtrate, charged into an automatic liquid extractor, wa extracted with ether over 36 to 48 hours. An ether fraction was recovered, and the ether was removed under reduced pressure. The remaining part was further concentrated whereby crude crystal of fi-cis-succinic acid of Gauche form was yielded. This crude crystal had yellow color and characteristic fermentative odor. From one liter of culture solution, about one gram of crude crystal wa obtained.

The crude crystal thus obtained was dissolved into a small amount of hot ether, and filtrate with small amount of carbon powder, and then cooled in order to recrystallization. Repeating recrystallization, about 500 mg. of pure crystal were obtained from about one gram of the crude one.

Similarly fl-cis-succinic acid of Gauche form was extracted from a culture solution which was obtained by cultivation of the other strains of Saccharomyces genus than Saach. steineri var. hara such as referred to above. In this case, to 400 mg. of pure crystal was recovered from one liter of the culture solution.

EXTRACTION OF AN ENZYME LIKE SUBST. CON- TAINING LIQUOR FROM THE CULTURE SOLU- TION From the culture solution, mycelia were removed by filtration. By means of an automatic liquid extractor, the filtrate was extracted with ether for 36 to 48 hours. An ether fraction was recovered, and the ether was removed under reduced pressure. The remaining part was a brown colored liquid containing /3-cis-succinic acid of Gauche form together with the yeast used. This liquor (including the said yellow substance) was recovered is an amount of about 5 cc. from One liter of the culture solution.

SYNTHETIC PROCEDURES USING THE ENZYME- LIKE YELLOW SUBSTANCE-CONTAINING LIQUOR A mixture of equal amounts of known, commercially available succinic acid and maleic acid (or maleic anhydride) was dissolved in twice amount of distilled water or of less than 50% ethanol solution with heat. After cooling below 50 C., the resulting solution was combined with the above extracted enzyme like yellow substance which was in such amount as corresponding to more than 20% by weight of the said solution. While shielding from day light, the resulting mixture was concentrated under reduced pressure of less than 20 mm. Hg, whereby crude crystal of 8-cissuccinic acid of Gauche form was yielded The crude crystal could be recrystallized in the same manner mentioned above.

It should be noted that any other solvent than ether can not be used for the recovery of Gauche form cis-succinic acid from the culture solution because such solvent can not dissolve selectively the desired substances, and in the present, therefore, ether should be critically used for the extraction of the substances.

The cis-succinic acid of Gauche form of the present invention is characterized by the physical, chemical and physiological properties set forth in the following paragraphs.

Melting point.Melting points of the present compound and the commercial succinic acid were determined macroscopically as follows:

Sample was sealed in a capillary and heated comparatively lapid in sulfuric acid bath.

M.P. C. The present compound (ti-form) 183-184 Succinic acid, commercially available -]86 Mixed sample of the about two 184-185 Then a melting point of the present compound was determined by micro-method, and the following changes were observed:

Room temp. (hexagonal board or short cylinder shaped crystal):

- Snblimation point (147-156 C.) (crystal shape unchanged; B-form) 156-157 C. or above (needle-shaped crystal; a-form) Melting (196-197 C.)

-e (anhydride: B-type crystal) 147 0. 156 0.

156 C. (or-type crystal) (melt at 196197 0. Commercial succinic acid (,B-type crystal) don't subhne cool (melt at 186 C.) (at-type crystal DETERMINATION OF MOLECULAR WEIGHT Molecular weight of the substance was determined according to Akiya and Bergers method. Alcohol was used as solvent and azobenzene as known control. As a result, it was found that the molecular weight of the substance was approximately 120.

ELEMENTARY ANALYSIS Elementary analysis of this substance showed that the substance contained 40.83% of carbon, 5.14% of hydrogen and 54.03% of oxygen, but not nitrogen and halogen. See Table 6.

TABLE 6.-ELEMENTARY ANALYSIS Percent N C H cc. percent 4 The present substance 40.83 5.14 0 0 Succinic acid, commercially available--. 40.68 5.12 0 0 Maleic acid 41.39 3.47 0 0 ACID TIT RATION AND THE CURVE THEREOF An aqueous solution of this substance was titrated with N/lO-NaOH aqueous solution, and as its result the molecular weight was found to be about 119. Further such titration was repeated using an electric automatic titration apparatus, and the data were plotted on a titration curve. The present substance gave the similar titration curve to that of known succinic acid, and was found to be dicarboxylic acid (see FIG. 1).

QUALITATIVE TEST OF CARBONYL GROUP The precipitation reaction using 2,4-dinitrophenyl hydrazine was carried out for determining whether carbonyl group was present or not in the substance. It was found that the present substance had not C=O group.

QUALITATIVE TEST OF HYDROXYL GROUP As the result of diazo color reaction, it was found that the present substance did not contain hydroxyl group,

QUALITATIVE TEST OF ETHYLENICAL DOUBLE BOND The decolorization reaction of an aqueous solution of this substance by a potassium permanganate aqueous solution, and the decolorization reaction of a glacial acetic acid solution of this substance by a glacial acetic acid solution of bromine were tested, and in both cases decolorization was not observed. It was found that this substance had not an ethylenical double bond (C=C).

SOLUBILITY TO VARIOUS ORGANIC SOLVENTS This substance has a relatively lower solubility in water, ethanol or ether, than that of a known, commercially available succinic acid. Also this substance is slightly soluble in benzene and chloroform, and unsoluble in petroleum ether and carbon tetrachloride.

Solvents Chloro- Substances Water Ethanol Methanol Ether form Present substance- 1 25. 0 20. 0 10. 0 200 Non Succinic acid 13. 0 18. 5 6. 3 113 Non Maleic acid- Furmaric acid 158. 7 52. 0 32. 0 N011 1 Milliliter. 2 Freely. 3 Slightly.

One gram of samples were dissolved at 20 C. and one atmosphere pressure.

SPECIFIC ROTATION This substance (ti-form) and the polymorph thereof and a known, commercially available succinic acid were measured of their specific rotation:

(at) D, degrees The present substance (ii-form) (1% aq. soln.) 18.1 The present substance (a-form) (1% aq. soln.) 18.4 Commercial available succinic acid, (1% aq. soln.) 1.7

The brucine salt of this substance reacted at room temperature and that of a known succinic acid were measured of their specific rotation.

(at) D, degrees Brucine salt of this substance (1% aq. soln.) --19.3 Brucine succinate commercially available (1% aq.

soln.) 32.3

ULTRAVIOLET ABSORPTION In this test, an aqueous solution of this substance (1 mg./ 10 cc.) was used as sample. For contrast, known succinic acid, maleic acid and fuarnaric acid were employed. As shown in FIG. 2, the ultraviolet absorption spectrum of the substance shows only one maximum at 200 111,44, while those of succinic, maleic and fumaric acids show one maximum at 210 III/.t, 212 mp. and 208 m respectively.

INFRARED ABSORPTION The infrared absorption spectrum of this substance is entirely similar to that of a known, commercially available succinic acid, as seen in FIG. 3.

ANHYDRIDE OF B-CIS-SUCCINIC ACID OF GAUCHE FORM The substance (B-cis-succinic acid of Gauche form) is completely sublimated at temperatures between 147 and 156 C. thereby to convert it into its anhydrous form. Such anhydrous substance is characterized in that the infrared absorption spectrum thereof shows absorption bands at wavelength: 1780 and 1875 mr- This anhydrous substance when dissolved into water, immediately is converted into dicarboxylic acid by ring opening.

REACTIVITY TEST WITH OTHER COMPOUNDS When a 1 N aqueous solution of this substance is added with a 1 N ammonia solution, a chemical reaction takes place immediately at room temperature whereby asparagine and asparatic acid are formed. Commercially available succinic acid does not react with ammonia at a room temperature.

Rf VALUES ON PAPER CHROMATOGRAM The following data show the R values of the substance and other various known organic acids, which are determined by one dimensional paper-chromatogram.

TABLE 7 Rrvaluo Run No. 1 No. 2 No. 3

Acid:

This substance:

fl-form 0. 32 0. 45 0. 26 a-tornL 0. 31 0. 44 0. 26 Succiuie acid, commercially available.. 0. 34 0.50 0. 27 Fumaric acid 0. 37 0. 16 0. 27 Maloio acid 0. 31 0.44 0. 26 Gallic acid 0. 24 0. 24 0. l6 Malonic acid 0.16 0. 39 0. 13

The developing solvents used as follows:

Run No. 1:

Ethanol 80 Ammonia water 4 Water 16 Run No. 2:

Phenol 80 Water 20 Run No. 3:

n-Propanol 70 Ammonia water 30 In each of runs, the development was carried out at 20 C. for 15 hours, using Toyo filter paper No. 50 or 51 and bromo cresol green as coloring agent.

PHYSIOLOGICAL ACTIVITY OF THIS SUBSTANCE All concentrations of cis-succinic acid are in percentage by weight, unless otherwise indicated.

When fresh fish meat, whale meat, bird and animal meat, and the internal organs thereof are immersed into a 0.1 to 0.05% aqueous solution of this substance, the peculiar offensive odor disappears after immersion at an ordinary temperature for 2 to 6 hours or at to C. for 24 to 96 hours.

Fish or animal meat is digested with proteinase, and then indigestible residue and fat and fatty oils are removed therefrom. The resulting solution is adjusted with a 5 N KOH aqueous solution at a pH of 7.0 to 7.4, and this substance is added in an amount of 0.1 to 0.05% to them. While maintained at 70 to 100 C. for 60 minutes, the solution is readjusted at a pH of 7.6 to 8.0. After filtration, the filtrate is adjusted at a pH of 5.0 with 5 N HCl, and then concentrated to about one-fifth volume. The concentrate is added with about 1 or 2% starch, and then sprayor freeze-dried, whereby a deodorized powder containing a large amount of essential amino acids is obtained. Such powder is suited for the use of food.

A crude liquor removed of sodium gultamate crystals is adjusted at a pH of 7.0 to 7.2 by addition of 5 N KOH, and then heated up to 70 C. The liquor is added with 0.1 to 0.05% of the present substance, allowed to stand for 30 to 60 minutes, and then blown with sterile air for to 20 minutes. Volatile, odoriferous materials mainly in cluding dimethyl sulfite are liberated and purged away. The liquor thus treated is adjusted again at a pH of 7.6 to 8.0 with a KOH aqueous solution and then filtered. Black sediment is removed. The filtrate is adjusted at a pH of 4.6 to 4.7 with 5 N HCl. Thus a deodorized solution removed of sodium glutamate crystals (or amino acids) is obtained. This liquid is quite suitable to soy-sauce additive.

Mucous odor and putrefied tissue odor are removed by washing the area with a 0.1-0.05% aqueous solution of Amine Aspamtic acid CO0H COOH Amine Asparagin type Amine That is, when meat or fish is hydrolyzed, various kinds of amino acids can be produced, but the hydrolysis solution may contain various amines as a by-product, which causes a characteristic odor of the meat or fish.

The Gauche form succinic acid can be easily reacted with these free amines at a room temperature to give aspartic acid type-or asparagin type products thereby inhibiting a characteristic deodorizing action. Such an action can not be found in the commercially available succinic acid.

What is claimed is:

1. A process for deodorizing amine-containing material comprising contacting the material with cissuccinic acid of Gauche form.

2. A process for deodorizing free amino-containing material comprising adding potassium hydroxide to a solution of said amine-containing material to adjust the pH of said solution to between 7.0 and 7.2, and adding to the adjusted solution about 0.05% to 0.1% of by weight of cissuccinic acid of Gauche form.

3. A process for deodorizing amine-containing material comprising contacting said amine containing material with an aqueous solution containing 0.05 to 0.1% by weight of cis-succinic'acid of Gauche form.

References Cited UNITED STATES PATENTS 2,972,542 2/1961 Levin 99l1l XR 3,113,104 12/1963 Bersworth 2155 XR 3,198,251 8/1965 Shore 2l55 XR 3,413,218 11/1968 Einsel 21-55 XR MORRIS O. WOLK, Primary Examiner BARRY S. RICHMAN, Assistant Examiner US. Cl. X.R. 

1. A PROCESS FOR DEODORIZING AMINE-CONTAINING MATERIAL COMPRISING CONTACTING THE MATERIAL WITH CIS-SUCCINIC ACID OF GAUCHE FORM. 